Methods of treating non-alcoholic steatohepatitis (nash)  using cysteamine products

ABSTRACT

The disclosure relates, in general, to treatment of fatty liver disorders comprising administering compositions comprising cysteamine products. The disclosure provides administration of enterically coated cysteamine compositions to treat fatty liver disorders, such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/608,134, filed Sep. 10, 2012 (Now U.S. Pat. No. 9,149,448), which isa continuation of U.S. application Ser. No. 13/183,990, filed Jul. 15,2011 (Now U.S. Pat. No. 8,263,662), which is a continuation of U.S.application Ser. No. 12/745,504, filed May 28, 2010 (Now U.S. Pat. No.7,994,226), which is a U.S. National Stage Application filed under 35U.S.C. §371 and claims priority to International Application No.PCT/US08/85064, filed Nov. 28, 2008, which application claims priorityof U.S. Provisional Appl. No. 60/991,517 filed Nov. 30, 2007, and U.S.Provisional Appl. No. 61/085,397 filed Jul. 31, 2008, the disclosures ofeach of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates in general to materials and methods to treat fattyliver disease using cysteamine products.

BACKGROUND

Fatty liver disease (or steatohepatis) is often associated withexcessive alcohol intake or obesity, but also has other causes such asmetabolic deficiencies including insulin resistance and diabetes. Fattyliver results from triglyceride fat accumulation in vacuoles of theliver cells resulting in decreased liver function, and possibly leadingto cirrhosis or hepatic cancer.

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum ofdisease occurring in the absence of alcohol abuse. A satisfactorytreatment for fatty liver disease, such as NAFLD and NASH is notpresently available.

SUMMARY

The disclosure provides a method of treating a subject suffering fromfatty liver disease comprising administering a therapeutically effectiveamount of a cysteamine composition. In one embodiment, the fatty liverdisease is selected from the group consisting of non-alcoholic fattyacid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fattyliver disease resulting from hepatitis, fatty liver disease resultingfrom obesity, fatty liver disease resulting from diabetes, fatty liverdisease resulting from insulin resistance, fatty liver disease resultingfrom hypertriglyceridemia, Abetalipoproteinemia, glycogen storagediseases, Weber-Christian disease, Wolmans disease, acute fatty liver ofpregnancy, and lipodystrophy. In another embodiment, the total dailydose of cysteamine composition is about 0.5-1.0 g/m². In yet anotherembodiment, the cysteamine composition is administered at a frequency of4 or less times per day (e.g., one, two or three times per day). In oneembodiment, the composition is a delayed or controlled release dosageform that provides increased delivery of the cysteamine or cysteaminederivative to the small intestine. The delay or controlled release formcan provide a C_(max) of the cysteamine or cysteamine derivative, or abiologically active metabolite thereof, that is at least about 35%, 50%,75% or higher than the C_(max) provided by an immediate release dosageform containing the same amount of the cysteamine or cysteaminederivative. In yet another embodiment, the delayed or controlled releasedosage form comprises an enteric coating that releases the cysteaminecomposition when the composition reaches the small intestine or a regionof the gastrointestinal tract of a subject in which the pH is greaterthan about pH 4.5. For example, the coating can be selected from thegroup consisting of polymerized gelatin, shellac, methacrylic acidcopolymer type C NF, cellulose butyrate phthalate, cellulose hydrogenphthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate(PVAP), cellulose acetate phthalate (CAP), cellulose acetatetrimellitate (CAT), hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulosesuccinate, carboxymethyl ethylcellulose (CMEC), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and acrylic acid polymersand copolymers, typically formed from methyl acrylate, ethyl acrylate,methyl methacrylate and/or ethyl methacrylate with copolymers of acrylicand methacrylic acid esters. The composition can be administered orallyor parenterally. In another embodiment, the method results inimprovement in liver fibrosis compared to levels before administrationof the cysteamine composition. In yet another embodiment, the methodresults in a reduction in fat content of liver, a reduction in theincidence of or progression of cirrhosis, or a reduction in theincidence of hepatocellular carcinoma. In one embodiment, the methodresults in a decrease in hepatic aminotransferase levels compared tolevels before administration of the cysteamine composition. In a furtherembodiment, the administering results in a reduction in hepatictransaminase of between approximately 10% to 40% compared to levelsbefore treatment. In yet another embodiment, the administering resultsin a reduction in alanine or aspartate aminotransferase levels in atreated patient to approximately 30%, 20% or 10% above normal ALTlevels, or at normal ALT levels. In yet other embodiment, theadministering results in a reduction in serum ferritin levels comparedto levels before treatment with the cysteamine composition. The methodsand composition of the disclosure can also include administering asecond agent in combination with a cysteamine composition to treat fattyliver disease. The subject can be an adult, adolescent or child.

In one aspect, the disclosure provides a method of treating a patientsuffering from fatty liver disease, including NAFLD or NASH, comprisingadministering a therapeutically effective amount of a compositioncomprising a cysteamine product. The methods of the disclosure alsoinclude use of a cysteamine product in preparation of a medicament fortreatment of fatty liver disease, and use of a cysteamine product inpreparation of a medicament for administration in combination with asecond agent for treating fatty liver disease. Also included is use of asecond agent for treating fatty liver disease in preparation of amedicament for administration in combination with a cysteamine product.Further provided are kits comprising a cysteamine product for treatmentof fatty liver disease, optionally with a second agent for treatingfatty liver disease, and instructions for use in treatment of fattyliver disease. The term “fatty liver disease” may include or excludeNASH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of cysteamine treatment at 0, 75 and 250mg/kg/day, delivered intraperitoneally, on aspartate aminotransferase(AST) levels in animals fed a high fat diet (HFD) for 8 days. AST levelsfor control animals, not fed a HFD, are also shown. The graph depictsmean AST values from blood samples collected on study day-1 (“pre”) andon study day 8 (SD8).

FIG. 2 shows the effect of cysteamine treatment at 0, 75 and 250mg/kg/day, delivered intraperitoneally, on cholesterol levels in animalsfed a HFD for 8 days. Cholesterol levels for control animals, not fed aHFD, are also shown. The graph depicts mean cholesterol values fromblood samples collected on study day-1 (“pre”) and on study day 8 (SD8).

FIG. 3 shows the effect of cysteamine treatment at 0, 75 and 250mg/kg/day, delivered intraperitoneally, on low densitylipoprotein-cholesterol (LDL-cholesterol) levels in animals fed a HFDfor 8 days. LDL-cholesterol levels for control animals, not fed a HFD,are also shown. The graph depicts mean LDL-cholesterol values from bloodsamples collected on study day-1 (“pre”) and on study day 8 (SD8).

FIG. 4 shows the effect of cysteamine treatment at 0, 75 and 250mg/kg/day, delivered intraperitoneally, on lactate dehydrogenase (LDH)levels in animals fed a HFD for 8 days. LDH levels for control animals,not fed a HFD, are also shown. The graph depicts mean LDH values fromblood samples collected on study day −1 (“pre”) and on study day 8(SD8).

FIG. 5 shows the effect of cysteamine treatment at target doses of 0,25, 75 and 250 mg/kg/day, delivered via drinking water, on AST levels inanimals fed a HFD for 8 weeks. AST levels for control animals, not fed aHFD, are also shown. The graph depicts mean AST values±SEM from bloodsamples collected on study day-1 (“week 0”) and on the last day of theweek indicated (week 2, 4, 6 or 8).

FIG. 6 shows the effect of cysteamine treatment at target doses of 0,25, 75 and 250 mg/kg/day, delivered via drinking water, on LDH levels inanimals fed a HFD for 8 weeks. LDH levels for control animals, not fed aHFD, are also shown. The graph depicts mean LDH values±SEM from bloodsamples collected on study day-1 (“week 0”) and on the last day of theweek indicated (week 2, 4, 6 or 8).

FIG. 7 shows the effect of cysteamine treatment at target doses of 0,25, 75 and 250 mg/kg/day, delivered via drinking water, on high densitylipoprotein cholesterol (HDL-cholesterol) levels in animals fed a HFDfor 8 weeks. HDL-cholesterol levels for control animals, not fed a HFD,are also shown. The graph depicts mean HDL-cholesterol values±SEM fromblood samples collected on study day-1 (“week 0”) and on the last day ofthe week indicated (week 2, 4, 6 or 8).

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a derivative”includes a plurality of such derivatives and reference to “a subject”includes reference to one or more subjects and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The disclosure provides new therapeutics that can alleviate the symptomsassociated with fatty liver disease in patients suffering from thedisease. The disclosure provides cysteamine compositions which providean effective therapy for patients in need of treatment.

The following references provide one of skill with a general definitionof many of the terms used in this disclosure: Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THECAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THEGLOSSARY OF GENETICS, 5TH ED., R. Rieger, et al. (eds.), Springer Verlag(1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY(1991).

Cysteamine is a precursor to the protein glutathione (GSH) precursor,and is currently FDA approved for use in the treatment of cystinosis, anintra-lysosomal cystine storage disorder. In cystinosis, cysteamine actsby converting cystine to cysteine and cysteine-cysteamine mixeddisulfide which are then both able to leave the lysosome through thecysteine and lysine transporters respectively (Gahl et al., N Engl J Med2002; 347(2):111-21). Within the cytosol the mixed disulfide can bereduced by its reaction with glutathione and the cysteine released canbe used for further GSH synthesis. The synthesis of GSH from cysteine iscatalyzed by two enzymes, gamma-glutamylcysteine synthetase and GSHsynthetase. This pathway occurs in almost all cell types, with the liverbeing the major producer and exporter of GSH. The reducedcysteine-cysteamine mixed disulfide will also release cysteamine, which,in theory is then able to re-enter the lysosome, bind more cystine andrepeat the process (Dohil et al., J Pediatr 2006; 148(6):764-9). In arecent study in children with cystinosis, enteral administration ofcysteamine resulted in increased plasma cysteamine levels, whichsubsequently caused prolonged efficacy in the lowering of leukocytecystine levels (Dohil et al., J Pediatr 2006; 148(6):764-9). This mayhave been due to “re-cycling” of cysteamine when adequate amounts ofdrug reached the lysosome. If cysteamine acts in this fashion, then GSHproduction may also be significantly enhanced.

Cysteamine is a potent gastric acid-secretagogue that has been used inlaboratory animals to induce duodenal ulceration; studies in humans andanimals have shown that cysteamine-induced gastric acid hypersecretionis most likely mediated through hypergastrinemia. In previous studiesperformed in children with cystinosis who suffered regular uppergastrointestinal symptoms, a single oral dose of cysteamine (11-23mg/kg) was shown to cause hypergastrinemia and a 2 to 3-fold rise ingastric acid-hypersecretion, and a 50% rise in serum gastrin levels.Symptoms suffered by these individuals included abdominal pain,heartburn, nausea, vomiting, and anorexia. U.S. patent application Ser.No. 11/990,869 and published International Publication No. WO2007/089670, both claiming priority to U.S. Provisional Patentapplication No. 60/762,715, filed Jan. 26, 2006, (all of which areincorporated by reference herein in their entirety) showed thatcysteamine induced hypergastrinemia arises, in part, as a local effecton the gastric antral-predominant G-cells in susceptible individuals.The data also suggest that this is also a systemic effect of gastrinrelease by cysteamine. Depending on the route of administration, plasmagastrin levels usually peak after intragastric delivery within 30minutes whereas the plasma cysteamine levels peak later.

Subjects with cystinosis are required to ingest oral cysteamine(CYSTAGON®) every 6 hours day and night. When taken regularly,cysteamine can deplete intracellular cystine by up to 90% (as measuredin circulating white blood cells), and this had been shown to reduce therate of progression to kidney failure/transplantation and also toobviate the need for thyroid replacement therapy. Because of thedifficulty in taking CYSTAGON®, reducing the required dosing improvesthe adherence to therapeutic regimen. International Publication No. WO2007/089670 demonstrates that delivery of cysteamine to the smallintestine reduces gastric distress and ulceration, increases C_(max) andincreases AUC. Delivery of cysteamine into the small intestine is usefuldue to improved absorption rates from the small intestine, and/or lesscysteamine undergoing hepatic first pass elimination when absorbedthrough the small intestine. A decrease in leukocyte cystine wasobserved within an hour of treatment.

The disclosure provides cysteamine products useful in the treatment offatty liver diseases and disorders. A cysteamine product refers,generally, to cysteamine, cystamine, or a biologically active metabolitethereof, or combination of cysteamine or cystamine, and includescysteamine or cystamine salts, esters, amides, alkylated compounds,prodrugs, analogs, phosphorylated compounds, sulfated compounds, orother chemically modified forms thereof, by such techniques as labeling(e.g., with radionuclides or various enzymes), or covalent polymerattachment such as pegylation (derivatization with polyethylene glycol).

A cysteamine product includes cysteamine, cystamine, biologically activemetabolites, chemically modified forms of the compound, by suchtechniques as esterification, alkylation (e.g., C1, C2 or C3), labeling(e.g., with radionuclides or various enzymes), covalent polymerattachment such as pegylation (derivatization with polyethylene glycol)or mixtures thereof. In some embodiments, cysteamine products include,but are not limited to, hydrochloride salts, bitartrate salts,phosphorylated derivatives, and sulfated derivatives. Examples of othercysteamine products include 2-aminopropane thiol-1, 1-aminopropanethiol-2, N- and S-substituted cysteamine, AET, aminoalkyl derivatives,phosphorothioate, amifostine (U.S. Pat. No. 4,816,482). In oneembodiment, a cysteamine product specifically excludes N-acetylcysteine.In one embodiment, cysteamine products comprise, but are not limited to,structures described below:

wherein n represents 2 or 3, R₁ and R₂ each represents a hydrogen atom,or an alkyl group optionally substituted by a hydroxy, amino, alkylaminoor dialkylamino group, or represents a cycloalkyl or aryl group, and X₁represents a group selected from the group consisting of ═N—CN, ═N—NO₂,═N—COR₃, ═N—NR—COOR₃, ═N—NR—CONH₂, ═N—SO₂R₃, ═CH—NO₂, —CH—SO₂R₃,═C(CN)₂, ═C(CN)COOR₃ and ═C(CN)CONH₂, wherein R₃ is an alkyl or arylgroup. In another aspect, a cysteamine product can comprise a cysteamineradical linked to any number of non-toxic groups as set forth below:

wherein R₁ represents hydrogen atom or a straight chain or a branchedalkyl group having 1 to 10 carbon atoms.

Pharmaceutically acceptable salts of cysteamine products are alsoincluded and comprise pharmaceutically-acceptable anions and/or cations.Pharmaceutically-acceptable cations include among others, alkali metalcations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metal cations (e.g., Ca²⁺,Mg²⁺), non-toxic heavy metal cations and ammonium (NH⁴⁺) and substitutedammonium (N(R′)⁴⁺, where R′ is hydrogen, alkyl, or substituted alkyl,i.e., including, methyl, ethyl, or hydroxyethyl, specifically, trimethylammonium, triethyl ammonium, and triethanol ammonium cations).Pharmaceutically-acceptable anions include among other halides (e.g.,Cl⁻, Br⁻), sulfate, acetates (e.g., acetate, trifluoroacetate),ascorbates, aspartates, benzoates, citrates, and lactate.

Cysteamine products can be enterically coated. An enterically coateddrug or tablet refers, generally, to a drug or tablet that is coatedwith a substance (an “enteric coating”) that remains intact orsubstantially intact such that the drug or tablet is passed through thestomach but dissolves and releases the drug in the small intestine.

An enteric coating can be a polymer material or materials which encase amedicament core (e.g., cystamine, cysteamine, CYSTAGON® or othercysteamine product). Typically a substantial amount or all of theenteric coating material is dissolved before the medicament ortherapeutically active agent is released from the dosage form, so as toachieve delayed dissolution or delivery of the medicament core. Asuitable pH-sensitive polymer is one which will dissolve in intestinalenvironment at a higher pH level (pH greater than 4.5), such as withinthe small intestine and therefore permit release of thepharmacologically active substance in the regions of the small intestineand not in the upper portion of the GI tract, such as the stomach.

The cysteamine product may also include additional pharmaceuticallyacceptable carriers or vehicles. A pharmaceutically acceptable carrieror vehicle refers, generally, to materials that are suitable foradministration to a subject wherein the carrier or vehicle is notbiologically harmful, or otherwise, cause undesirable effects. Suchcarriers or vehicles are typically inert ingredients of a medicament.Typically a carrier or vehicle is administered to a subject along withan active ingredient without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof a pharmaceutical composition in which it is contained.

A cyteamine product or other active ingredient can comprise apharmaceutically acceptable salt, ester or other derivative. Forexample, salts, esters or other derivatives comprise biologically activeforms having a similar biological effect compared to a parent compound.Exemplary salts include hydrochloride salt and bistartrate salts.

An active ingredient, pharmaceutical or other composition of thedisclosure can comprise a stabilizing agent. Stabilizing agents,generally, refer to compounds that lower the rate at which apharmaceutical degrades, particularly an oral pharmaceutical formulationunder environmental conditions of storage.

As used herein, a “therapeutically effective amount” or “effectiveamount” refers to that amount of the compound sufficient to result inamelioration of symptoms, for example, treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions,typically providing a statistically significant improvement in thetreated patient population. When referencing an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When referring to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, including serially or simultaneously. In one embodiment,a therapeutically effective amount of the cysteamine product amelioratessymptoms, including but not limited to, liver fibrosis, fat content ofliver, incidence of or progression of cirrhosis, incidence ofhepatocellular carcinoma, increased hepatic aminotransferase levels,such as ALT and AST, increased serum ferritin, elevated levels ofgamma-glutamyltransferase (gamma-GT), and elevated levels of plasmainsulin, cholesterol and triglyceride.

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum ofdisease occurring in the absence of alcohol abuse. It is characterizedby the presence of steatosis (fat in the liver) and may represent ahepatic manifestation of the metabolic syndrome (including obesity,diabetes and hypertriglyceridemia). NAFLD is linked to insulinresistance, it causes liver disease in adults and children and mayultimately lead to cirrhosis (Skelly et al., J Hepatol 2001; 35: 195-9;Chitturi et al., Hepatology 2002; 35(2):373-9). The severity of NAFLDranges from the relatively benign isolated predominantly macrovesicularsteatosis (i.e., nonalcoholic fatty liver or NAFL) to non-alcoholicsteatohepatitis (NASH) (Angulo et al., J Gastroenterol Hepatol 2002; 17Suppl:S186-90). NASH is characterized by the histologic presence ofsteatosis, cytological ballooning, scattered inflammation andpericellular fibrosis (Contos et al., Adv Anat Pathol 2002; 9:37-51).Hepatic fibrosis resulting from NASH may progress to cirrhosis of theliver or liver failure, and in some instances may lead to hepatocellularcarcinoma.

The degree of insulin resistance (and hyperinsulinemia) correlates withthe severity of NAFLD, being more pronounced in patients with NASH thanwith simple fatty liver (Sanyal et al., Gastroenterology 2001;120(5):1183-92). As a result, insulin-mediated suppression of lipolysisoccurs and levels of circulating fatty acids increase. Two factorsassociated with NASH include insulin resistance and increased deliveryof free fatty acids to the liver. Insulin blocks mitochondrial fattyacid oxidation. The increased generation of free fatty acids for hepaticre-esterification and oxidation results in accumulation of intrahepaticfat and increases the liver's vulnerability to secondary insults.

Glutathione (gammaglutamyl-cysteinyl-glycine; GSH) is a major endogenousantioxidant and its depletion is implicated in the development ofhepatocellular injury (Wu et al., J Nutr 2004; 134(3):489-92). One suchinjury is acetaminophen poisoning, where reduced GSH levels becomedepleted in an attempt to conjugate and inactivate the hepatotoxicmetabolite of the drug. After a toxic dose of acetaminophen, excessmetabolite (N-acetyl-benzoquinoneimine) covalently binds to hepaticproteins and enzymes resulting in liver damage (Wu et al., J Nutr 2004;134(3):489-92; Prescott et al., Annu Rev Pharmacol Toxicol 1983;23:87-101). Increased glutathione levels appears therefore to have someprotective effects through the reduction of ROS. Glutathione itself isdoes not enter easily into cells, even when given in large amounts.However, glutathione precursors do enter into cells and some GSHprecursors such as N-acetylcysteine have been shown to be effective inthe treatment of conditions such as acetaminophen toxicity by slowing orpreventing GSH depletion (Prescott et al., Annu Rev Pharmacol Toxicol1983; 23:87-101). Examples of GSH precursors include cysteine,N-acetylcysteine, methionine and other sulphur-containing compounds suchas cysteamine (Prescott et al., J Int Med Res 1976; 4(4 Suppl):112-7).

Cysteine is a major limiting factor for GSH synthesis and that factors(e.g., insulin and growth factors) that stimulate cysteine uptake bycells generally result in increased intracellular GSH levels (Lyons etal., Proc Natl Acad Sci USA 2000; 97(10):5071-6; Lu S C. Curr Top CellRegul 2000; 36:95-11).

N-acetylcysteine has been administered to patients with NASH. In reportsfrom Turkey, obese individuals with NASH treated with N-acetylcysteinefor 4-12 weeks exhibited an improvement in aminotransferase levels andgamma-GT even though there was no reported change in subject body massindex (Pamuk et al., J Gastroenterol Hepatol 2003; 18(10):1220-1).

WA Cysteamine (HS—CH₂—CH₂—NH₂) is able to cross cell membranes easilydue to its small size. At present, cysteamine is FDA-approved only forthe treatment of cystinosis, an intra-lysosomal cystine storagedisorder. In cystinosis, cysteamine acts by converting cystine tocysteine and cysteine-cysteamine mixed disulfide which are then bothable to leave the lysosome through the cysteine and lysine transportersrespectively (Gahl et al., N Engl J Med 2002; 347(2):111-21). Treatmentwith cysteamine has been shown to result in lowering of intracellularcystine levels in circulating leukocytes (Dohil et al., J. Pediatr 2006;148(6):764-9).

Studies in mice and humans showed cysteamine to be effective inpreventing acetaminophen-induced hepatocellular injury (Prescott et al.,Lancet 1972; 2(7778):652; Prescott et al., Br Med J 1978; 1(6116):856-7;Mitchell et al., Clin Pharmacol Ther 1974; 16(4):676-84). Cystamine andcysteine have been reported to reduce liver cell necrosis induced byseveral hepatotoxins. (Toxicol Appl Pharmacol. 1979 April; 48(2):221-8).Cystamine has been shown to ameliorate liver fibrosis induced by carbontetrachloride via inhibition of tissue transglutaminase (Qiu et al.,World J Gastroenterol. 13:4328-32, 2007).

The prevalence of NAFLD in children is unknown because of therequirement of histologic analysis of liver in order to confirm thediagnosis (Schwimmer et al., Pediatrics 2006; 118(4):1388-93). However,estimates of prevalence can be inferred from pediatric obesity datausing hepatic ultra-sonongraphy and elevated serum transaminase levelsand the knowledge that 85% of children with NAFLD are obese. Data fromthe National Health and Nutrition Examination Survey has revealed athreefold rise in the prevalence of childhood and adolescent obesityover the past 35 years; data from 2000 suggests that 14-16% childrenbetween 6-19 yrs age are obese with a BMI>95% (Fishbein et al., JPediatr Gastroenterol Nutr 2003; 36(1):54-61), and also that fact that85% of children with NAFLD are obese.

In patients with histologically proven NAFLD, serum hepaticaminotransferases, specifically alanine aminotransferase (ALT), levelsare elevated from the upper limit of normal to 10 times this level(Schwimmer et al., J Pediatr 2003; 143(4):500-5; Rashid et al., JPediatr Gastroenterol Nutr 2000; 30(1):48-53). The ratio of ALT/AST(aspartate aminotransferase) is >1 (range 1.5-1.7) which differs fromalcoholic steatohepatitis where the ratio is generally <1. Otherabnormal serologic tests that may be abnormally elevated in NASH includegamma-glutamyltransferase (gamma-GT) and fasting levels of plasmainsulin, cholesterol and triglyceride.

The exact mechanism by which NAFLD develops into NASH remains unclear.Because insulin resistance is associated with both NAFLD and NASH, it ispostulated that other additional factors are also required for NASH toarise. This is referred to as the “two-hit” hypothesis (Day CP. BestPract Res Clin Gastroenterol 2002; 16(5):663-78) and involves, firstly,an accumulation of fat within the liver and, secondly, the presence oflarge amounts of free radicals with increased oxidative stress.Macrovesicular steatosis represents hepatic accumulation oftriglycerides, and this in turn is due to an imbalance between thedelivery and utilization of free fatty acids to the liver. Duringperiods of increased calorie intake, triglyceride will accumulate andact as a reserve energy source. When dietary calories are insufficient,stored triglycerides (in adipose) undergo lipolysis and fatty acids arereleased into the circulation and are taken up by the liver. Oxidationof fatty acids will yield energy for utilization. Treatment of NASHcurrently revolves around the reduction of the two main pathogeneticfactors, namely, fat accumulation within the liver and excessiveaccumulation of free radicals causing oxidative stress. Fat accumulationis diminished by reducing fat intake as well as increasing caloricexpenditure. One therapeutic approach is sustained and steady weightloss. Although not definitively proven, a >10% loss in body weight hasbeen shown in some cases to reduce hepatic fat accumulation, normalizeliver transaminases and improve hepatic inflammation and fibrosis (Uenoet al., J Hepatol 1997; 27(1):103-7; Vajro et al., J Pediatr 1994;125(2):239-41; Franzese et al., Dig Dis Sci 1997; 42(7):1428-32).

Reduction of oxidative stress through treatment with antioxidants hasalso been shown to be effective in some studies. For example, obesechildren who had steatosis were treated with vitamin E (400-1000 IU/day)for 4-10 months (Lavine J Pediatr 2000; 136(6):734-8). Despite anysignificant change in BMI, the mean ALT levels decreased from 175±106IU/L to 40±26 IU/L (P<0.01) and mean AST levels decreased from 104±61IU/L to 33±11 IU/L (P<0.002). Hepatic transaminases increased in thosepatients who elected to discontinue vitamin E therapy. An adult studyusing vitamin E for one year demonstrated similar reduction of hepatictransaminases as well as the fibrosis marker TGFβ levels (Hasegawa etal., Aliment Pharmacol Ther 2001; 15(10):1667-72).

Steatosis also may develop into steatohepatitis through oxidative stressdue to reactive oxygen species (ROS) and decreased anti-oxidant defense(Sanyal et al., Gastroenterology 2001; 120(5):1183-92). ROS can begenerated in the liver through several pathways including mitochondria,peroxisomes, cytochrome P450, NADPH oxidase and lipooxygenase (Sanyal etal., Nat Clin Pract Gastroenterol Hepatol 2005; 2(1):46-53). Insulinresistance and hyperinsulinism has been shown to increase hepaticoxidative stress and lipid peroxidation through increased hepatic CYP2EIactivity (Robertson et al., Am J Physiol Gastrointest Liver Physiol2001; 281(5):G1135-9; Leclercq et al., J Clin Invest 2000;105(8):1067-75).

Currently, much of what is understood of the pathogenesis of NAFLD hasarisen from animal studies. A number of mouse models which exhibitsteatosis/steatohepatitis exist and include genetically alteredleptin-deficient (ob/ob) or leptin resistant (db/db) and the dietarymethionine/choline deficient (MCD) model. Studies comparing male andfemale rats of varying strains (Wistar, Sprague-Dawley, Long-Evans) witha mouse strain (C57BL/6) as models for NASH have been undertaken. Theseanimals were fed for 4 weeks with an MCD diet; although ALT elevationand steatosis were more noticeable in the Wistar rat, the overallhistologic changes in the liver of the mice were more constant withchanges due to NASH. More recently the use of supra-nutritional diets inanimals has resulted in a NAFLD model that physiologically moreresembles the human phenotype. The medical conditions most commonlyassociated with NAFLD are obesity, Type II diabetes and dyslipidemia.These conditions can be induced by feeding mice and rats with high fator sucrose diets. Rats fed with a >70% fat-rich diet for 3 weeksdeveloped pan-lobular steatosis, patchy inflammation, enhanced oxidativestress, and increased plasma insulin concentrations suggesting insulinresistance. NASH mice have been induced through intragastricoverfeeding. Mice were fed up to 85% in excess of their standard intakefor 9 weeks. The mice became obese with 71% increase in final bodyweight; they demonstrated increase white adipose tissue, hyperglycemia,hyperinsulinemia, hyperleptinemia, glucose intolerance and insulinresistance. Of these mice 46% developed increased ALT (121=/−27 vs13+/−1 U/L) as well as histologic features suggestive of NASH. Thelivers of the overfed mice were about twice as large expected, beige incolor with microscopic evidence of lipid droplets, cytoplasmic vacuolesand clusters of inflammation.

Mouse models of NASH are created through specific diets (methioninecholine deficient, MCD) or intragastric overfeeding. These mice developserologic and histologic features of NASH. NASH mice are useful inscreening and measuring the effects cysteamine on NASH related diseaseand disorders. For example, the effect of treatment can be measured byseparating the NASH mice into a control group where animals willcontinue to receive MCD diet only and three other treatment groups wheremice will receive MCD diet as well as anti-oxidant therapy. The threetherapy groups will receive cysteamine 50 mg/kg/day, 100 mg/kg/day andsAME.

Cysteamine is a small molecule (HS—CH2-CH2-NH2) which is able to crosscell membranes easily. Cysteamine is a potent gastric acid-secretagoguethat has been used in laboratory animals to induce duodenal ulceration;studies in humans and animals have shown that cysteamine-induced gastricacid hypersecretion is most likely mediated through hypergastrinemia.

In addition, sulfhydryl (SH) compounds such as cysteamine, cystamine,and glutathione are among the most important and active intracellularantioxidants. Cysteamine protects animals against bone marrow andgastrointestinal radiation syndromes. The rationale for the importanceof SH compounds is further supported by observations in mitotic cells.These are the most sensitive to radiation injury in terms of cellreproductive death and are noted to have the lowest level of SHcompounds. Conversely, S-phase cells, which are the most resistant toradiation injury using the same criteria, have demonstrated the highestlevels of inherent SH compounds. In addition, when mitotic cells weretreated with cysteamine, they became very resistant to radiation. It hasalso been noted that cysteamine may directly protect cells againstinduced mutations. The protection is thought to result from scavengingof free radicals, either directly or via release of protein-bound GSH.An enzyme that liberates cysteamine from coenzyme A has been reported inavian liver and hog kidney. Recently, studies have appeareddemonstrating a protective effect of cysteamine against the hepatotoxicagents acetaminophen, bromobenzene, and phalloidine.

Cystamine, in addition, to its role as a radioprotectant, has been foundto alleviate tremors and prolong life in mice with the gene mutation forHuntington's disease (HD). The drug may work by increasing the activityof proteins that protect nerve cells, or neurons, from degeneration.Cystamine appears to inactivate an enzyme called transglutaminase andthus results in a reduction of huntingtin protein (Nature Medicine 8,143-149, 2002). In addition, cystamine was found to increase the levelsof certain neuroprotective proteins. However, due to the current methodsand formulation of delivery of cystamine, degradation and poor uptakerequire excessive dosing.

At present, cysteamine is FDA approved only for the treatment ofcystinosis. Patients with cystinosis are normally required to takecysteamine every 6 hours. Ideally, an effective controlled-releasepreparation of cysteamine with perhaps twice daily administration wouldimprove the quality of life for these patients.

The disclosure is not limited with respect to a specific cysteamine orcystamine salt or ester or derivative; the compositions of thedisclosure can contain any cysteamine or cystamine, cysteamine orcystamine derivative, or combination of cysteamine or cystamines. Theactive agents in the composition, i.e., cysteamine or cystamine, may beadministered in the form of a pharmacologically acceptable salt, ester,amide, prodrug or analog or as a combination thereof. Salts, esters,amides, prodrugs and analogs of the active agents may be prepared usingstandard procedures known to those skilled in the art of syntheticorganic chemistry and described, for example, by J. March, “AdvancedOrganic Chemistry: Reactions, Mechanisms and Structure,” 4th Ed. (NewYork: Wiley-Interscience, 1992). For example, basic addition salts areprepared from the neutral drug using conventional means, involvingreaction of one or more of the active agent's free hydroxyl groups witha suitable base. Generally, the neutral form of the drug is dissolved ina polar organic solvent such as methanol or ethanol and the base isadded thereto. The resulting salt either precipitates or may be broughtout of solution by addition of a less polar solvent. Suitable bases forforming basic addition salts include, but are not limited to, inorganicbases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Preparation of estersinvolves functionalization of hydroxyl groups which may be presentwithin the molecular structure of the drug. The esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moietieswhich are derived from carboxylic acids of the formula R—COOH where R isalkyl, and typically is lower alkyl. Esters can be reconverted to thefree acids, if desired, by using conventional hydrogenolysis orhydrolysis procedures. Preparation of amides and prodrugs can be carriedout in an analogous manner. Other derivatives and analogs of the activeagents may be prepared using standard techniques known to those skilledin the art of synthetic organic chemistry, or may be deduced byreference to the pertinent literature.

The methods of compositions of the disclosure further provideenteric-coated compositions that result in less frequent dosing (2×/dayvs. 4×/day), increased patient compliance and fewer gastrointestinalside effects (e.g., pain, heartburn, acid production, vomiting) andother side effects (e.g., patients smell like rotten eggs—a particularcompliance problem as subjects reach puberty). The disclosure providesenteric-coated cysteamine compositions (sulfhydryl/CYSTAGON®) andcystamine compositions.

The disclosure provides methods for the treatment of fatty acid liverdisease, including, but not limited to non-alcoholic fatty acid liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liverdisease resulting from hepatitis, fatty liver disease resulting fromobesity, fatty liver disease resulting from diabetes, fatty liverdisease resulting from insulin resistance, fatty liver disease resultingfrom hypertriglyceridemia, Abetalipoproteinemia, glycogen storagediseases, Weber-Christian disease, Wolmans disease, acute fatty liver ofpregnancy, and lipodystrophy.

The effectiveness of a method or composition of the described herein canbe assessed, for example, by measuring leukocyte cystine concentrations.Additional measures of the efficacy of the methods of the disclosureinclude assessing relief of symptoms associated with fatty liver diseaseincluding, but not limited to, liver fibrosis, fat content of liver,incidence of or progression of cirrhosis, incidence of hepatocellularcarcinoma, elevated hepatic aminotransferase levels, increased alanineaminotransferase (ALT), increased aspartate aminotransferase (AST), andelevated serum ferritin. Dosage adjustment and therapy can be made by amedical specialist depending upon, for example, the severity of fattyliver disease and/or the concentration of cystine. For example,treatment of fatty liver disease may result in a reduction in hepatictransaminase of between approximately 10% to 40% compared to levelsbefore treatment. In a related embodiment, treatment results in areduction in alanine anminotransferase levels in a treated patient toapproximately 30%, 20% or 10% above normal ALT levels, or at normal ALTlevels (≧40 iu/L). In another embodiment, treatment with cysteamineproduct results in a reduction in aspartate anminotransferase levels ina patient to approximately 30%, 20% or 10% above normal AST levels orback to normal AST levels.

In one embodiment, the disclosure provides methods of treating NAFLusing cysteamine products through reducing the oxidative stress causedby reactive oxygen species (ROS) in steatohepatitis. Cysteamine canachieve this through its direct or indirect ability to enhanceglutathione levels within the liver. Glutathione has a protective effectagainst oxidative damage but itself does not enter easily into cells,even when given in large amounts treatment. Precursors of glutathionedo, however, enter into cells and include cysteine, N-acetylcyteine,s-adenosylmethionine (SAMe) and other sulphur-containing compounds suchas cysteamine.

The compositions of the disclosure can be used in combination with asecond agent or other therapies useful for treating NAFLD or NASH orother fatty acid liver disorders. For example, cysteamine productcompositions may be administered with drugs such asglitazones/thiazolidinediones that combat insulin resistance, includingmesylate (troglitazone (REZULIN®)), rosiglitazone (AVANDIA®),pioglitazone (ACTOS®), as well as other agents, including, but notlimited to, metformin, Sulfonylureas, Alpha-glucosidase inhibitors,Meglitinides, vitamin E, tetrahydrolipstatin (XENICAL™, milk thistleprotein (SILIPHOS®), and anti-virals.

Other therapies which reduce side effects of cysteamine products can becombined with the methods and compositions of the disclosure to treatdiseases and disorders that are attributed or result from NAFLD or NASH.Urinary phosphorus loss, for example, entails rickets, and it may benecessary to give a phosphorus supplement. Carnitine is lost in theurine and blood levels are low. Carnitine allows fat to be used by themuscles to provide energy. Hormone supplementation is sometimesnecessary. Sometimes the thyroid gland will not produce enough thyroidhormones. This is given as thyroxin (drops or tablets). Insulintreatment is sometimes necessary if diabetes appears, when the pancreasdoes not produce enough insulin. These treatments have become rarelynecessary in children whom are treated with cysteamine product, sincethe treatment protects the thyroid and the pancreas. Some adolescentboys require a testosterone treatment if puberty is late. Growth hormonetherapy may be indicated if growth is not sufficient despite a goodhydro electrolytes balance. Accordingly, such therapies can be combinedwith the cysteamine product compositions and methods of the disclosure.Additional therapies including the use of omeprazole (PRILOSEC®) canreduce adverse symptoms affecting the digestive tract.

The disclosure provides cysteamine products useful in the treatment offatty liver diseases and disorders. To administer cysteamine products ofthe disclosure to human or test animals, it is preferable to formulatethe cysteamine products in a composition comprising one or morepharmaceutically acceptable carriers. As set out above, pharmaceuticallyor pharmacologically acceptable carriers or vehicles refer to molecularentities and compositions that do not produce allergic, or other adversereactions when administered using routes well-known in the art, asdescribed below, or are approved by the U.S. Food and DrugAdministration or a counterpart foreign regulatory authority as anacceptable additive to orally or parenterally administeredpharmaceuticals. Pharmaceutically acceptable carriers include any andall clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like.

Pharmaceutical carriers include pharmaceutically acceptable salts,particularly where a basic or acidic group is present in a compound. Forexample, when an acidic substituent, such as —COOH, is present, theammonium, sodium, potassium, calcium and the like salts, arecontemplated for administration. Additionally, where an acid group ispresent, pharmaceutically acceptable esters of the compound (e.g.,methyl, tert-butyl, pivaloyloxymethyl, succinyl, and the like) arecontemplated as preferred forms of the compounds, such esters beingknown in the art for modifying solubility and/or hydrolysischaracteristics for use as sustained release or prodrug formulations.

When a basic group (such as amino or a basic heteroaryl radical, such aspyridyl) is present, then an acidic salt, such as hydrochloride,hydrobromide, acetate, maleate, pamoate, phosphate, methanesulfonate,p-toluenesulfonate, and the like, is contemplated as a form foradministration.

In addition, compounds may form solvates with water or common organicsolvents. Such solvates are contemplated as well.

The cysteamine product compositions may be administered orally,parenterally, transocularly, intranasally, transdermally,transmucosally, by inhalation spray, vaginally, rectally, or byintracranial injection. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intracisternalinjection, or infusion techniques. Administration by intravenous,intradermal, intramusclar, intramammary, intraperitoneal, intrathecal,retrobulbar, intrapulmonary injection and or surgical implantation at aparticular site is contemplated as well. Generally, compositions foradministration by any of the above methods are essentially free ofpyrogens, as well as other impurities that could be harmful to therecipient. Further, compositions for administration parenterally aresterile.

Pharmaceutical compositions of the disclosure containing a cysteamineproduct as an active ingredient may contain pharmaceutically acceptablecarriers or additives depending on the route of administration. Examplesof such carriers or additives include water, a pharmaceuticallyacceptable organic solvent, collagen, polyvinyl alcohol,polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulosesodium, polyacrylic sodium, sodium alginate, water-soluble dextran,carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose,xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin,propylene glycol, polyethylene glycol, Vaseline®, paraffin, stearylalcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol,lactose, a pharmaceutically acceptable surfactant and the like.Additives used are chosen from, but not limited to, the above orcombinations thereof, as appropriate, depending on the dosage form ofthe disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the cysteamine product to beadministered can be prepared in a physiologically acceptable vehicle orcarrier. For solutions or emulsions, suitable carriers include, forexample, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers.

A variety of aqueous carriers, e.g., water, buffered water, 0.4% saline,0.3% glycine, or aqueous suspensions may contain the active compound inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

In some embodiments, the cysteamine product of this disclosure can belyophilized for storage and reconstituted in a suitable carrier prior touse. Any suitable lyophilization and reconstitution techniques can beemployed. It is appreciated by those skilled in the art thatlyophilization and reconstitution can lead to varying degrees ofactivity loss and that use levels may have to be adjusted to compensate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

In one embodiment, the disclosure provides use of an enterically coatedcysteamine product composition. Enteric coatings prolong release untilthe cysteamine product reaches the intestinal tract, typically the smallintestine. Because of the enteric coatings, delivery to the smallintestine is improved thereby improving uptake of the active ingredientwhile reducing gastric side effects.

In some embodiments, the coating material is selected such that thetherapeutically active agent is released when the dosage form reachesthe small intestine or a region in which the pH is greater than pH 4.5.The coating may be a pH-sensitive material, which remain intact in thelower pH environs of the stomach, but which disintegrate or dissolve atthe pH commonly found in the small intestine of the patient. Forexample, the enteric coating material begins to dissolve in an aqueoussolution at pH between about 4.5 to about 5.5. For example, pH-sensitivematerials will not undergo significant dissolution until the dosage fromhas emptied from the stomach. The pH of the small intestine graduallyincreases from about 4.5 to about 6.5 in the duodenal bulb to about 7.2in the distal portions of the small intestine. In order to providepredictable dissolution corresponding to the small intestine transittime of about 3 hours (e.g., 2-3 hours) and permit reproducible releasetherein, the coating should begin to dissolve at the pH range within thesmall intestine. Therefore, the amount of enteric polymer coating shouldbe sufficient to substantially dissolved during the approximate threehour transit time within the small intestine, such as the proximal andmid-intestine.

Enteric coatings have been used for many years to arrest the release ofthe drug from orally ingestible dosage forms. Depending upon thecomposition and/or thickness, the enteric coatings are resistant tostomach acid for required periods of time before they begin todisintegrate and permit release of the drug in the lower stomach orupper part of the small intestines. Examples of some enteric coatingsare disclosed in U.S. Pat. No. 5,225,202 which is incorporated byreference fully herein. As set forth in U.S. Pat. No. 5,225,202, someexamples of coating previously employed are beeswax and glycerylmonostearate; beeswax, shellac and cellulose; and cetyl alcohol, masticand shellac, as well as shellac and stearic acid (U.S. Pat. No.2,809,918); polyvinyl acetate and ethyl cellulose (U.S. Pat. No.3,835,221); and neutral copolymer of polymethacrylic acid esters(Eudragit® L30D) (F. W. Goodhart et al., Pharm. Tech., pp. 64-71, April1984); copolymers of methacrylic acid and methacrylic acid methylester(Eudragit®), or a neutral copolymer of polymethacrylic acid esterscontaining metallic stearates (Mehta et al., U.S. Pat. Nos. 4,728,512and 4,794,001). Such coatings comprise mixtures of fats and fatty acids,shellac and shellac derivatives and the cellulose acid phthlates, e.g.,those having a free carboxyl content. See, Remington's at page 1590, andZeitova et al. (U.S. Pat. No. 4,432,966), for descriptions of suitableenteric coating compositions. Accordingly, increased adsorption in thesmall intestine due to enteric coatings of cysteamine productcompositions can result in improved efficacy.

Generally, the enteric coating comprises a polymeric material thatprevents cysteamine product release in the low pH environment of thestomach but that ionizes at a slightly higher pH, typically a pH of 4 or5, and thus dissolves sufficiently in the small intestines to graduallyrelease the active agent therein. Accordingly, among the most effectiveenteric coating materials are polyacids having a pKa in the range ofabout 3 to 5. Suitable enteric coating materials include, but are notlimited to, polymerized gelatin, shellac, methacrylic acid copolymertype C NF, cellulose butyrate phthalate, cellulose hydrogen phthalate,cellulose proprionate phthalate, polyvinyl acetate phthalate (PVAP),cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT),hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate, dioxypropyl methylcellulose succinate, carboxymethylethylcellulose (CMEC), hydroxypropyl methylcellulose acetate succinate(HPMCAS), and acrylic acid polymers and copolymers, typically formedfrom methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethylmethacrylate with copolymers of acrylic and methacrylic acid esters(Eudragit® NE, Eudragit® RL, Eudragit® RS). For example, the entericallycoating can comprise Eudragit® L30D, triethylcitrate, andhydroxypropylmethylcellulose (HPMC), wherein the coating comprises 10 to13% of the final product.

In one embodiment, the cysteamine product composition is administered intablet form. Tablets are manufactured by first enterically coating thecysteamine product. A method for forming tablets herein is by directcompression of the powders containing the enterically coated cysteamineproduct, optionally in combination with diluents, binders, lubricants,disintegrants, colorants, stabilizers or the like. As an alternative todirect compression, compressed tablets can be prepared usingwet-granulation or dry-granulation processes. Tablets may also be moldedrather than compressed, starting with a moist material containing asuitable water-soluble lubricant.

In some embodiments, the cysteamine product composition is a delayed orcontrolled release dosage form that provides a C_(max) of the cysteamineproduct that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, or 100% higher than the C_(max) provided by an immediaterelease dosage form containing the same amount of the cysteamineproduct. In some embodiments, the C_(max) is up to about 75%, 100%, 125%or 150% higher than the C_(max) of the immediate release dosage form.C_(max) refers to the maximum dose of the cysteamine product in theblood after dosing and provides an indicator that the drug is absorbedsystemically.

In some embodiments, the AUC of the delayed or controlled release dosageform is also increased by at least about 20%, 25%, 30%, 35%, 40%, 45%,or 50%, or up to about 50%, 60%, 75% or 100% relative to an immediaterelease dosage form. AUC or “area under the curve”, and refers to thekinetic curve derived when plasma drug concentration versus time ismeasured after dosing of a drug.

The preparation of delayed, controlled or sustained/extended releaseforms of pharmaceutical compositions with the desired pharmacokineticcharacteristics is known in the art and can be accomplished by a varietyof methods. For example, oral controlled delivery systems includedissolution-controlled release (e.g., encapsulation dissolution controlor matrix dissolution control), diffusion-controlled release (reservoirdevices or matrix devices), ion exchange resins, osmotic controlledrelease or gastroretentive systems. Dissolution controlled release canbe obtained, e.g., by slowing the dissolution rate of a drug in thegastrointestinal tract, incorporating the drug in an in soluble polymer,and coating drug particles or granules with polymeric materials ofvarying thickness. Diffusion controlled release can be obtained, e.g.,by controlling diffusion through a polymeric membrane or a polymericmatrix. Osmotically controlled release can be obtained, e.g., bycontrolling solvent influx across a semipermeable membrane, which inturn carries the drug outside through a laser-drilled orifice. Theosmotic and hydrostatic pressure differences on either side of themembrane govern fluid transport. Prolonged gastric retention may beachieved by, e.g., altering density of the formulations, bioadhesion tothe stomach lining, or increasing floating time in the stomach. Forfurther detail, see the Handbook of Pharmaceutical Controlled ReleaseTechnology, Wise, ed., Marcel Dekker, Inc., New York, N.Y. (2000),incorporated by reference herein in its entirety, e.g. Chapter 22 (“AnOverview of Controlled Release Systems”).

The concentration of cysteamine product in these formulations can varywidely, for example from less than about 0.5%, usually at or at leastabout 1% to as much as 15 or 20% by weight and are selected primarilybased on fluid volumes, manufacturing characteristics, viscosities,etc., in accordance with the particular mode of administration selected.Actual methods for preparing administrable compositions are known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

The cysteamine product is present in the composition in atherapeutically effective amount; typically, the composition is in unitdosage form. The amount of cysteamine product administered will, ofcourse, be dependent on the age, weight, and general condition of thesubject, the severity of the condition being treated, and the judgmentof the prescribing—physician. Suitable therapeutic amounts will be knownto those skilled in the art and/or are described in the pertinentreference texts and literature. Current non-enterically coated doses areabout 1.35 g/m² body surface area and are administered 4-5 times perday. In one aspect, the dose is administered either one time per day ormultiple times per day. The cysteamine product may be administered one,two or three or four or five times per day. In some embodiments, aneffective dosage of cysteamine product may be within the range of 0.01mg to 1000 mg per kg (mg/kg) of body weight per day. Further, theeffective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg,20 mg/kg/25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kgincrements up to 1000 mg/kg, or may range between any two of theforegoing values. In some embodiments, the cysteamine product isadministered at a total daily dose of from approximately 0.25 g/m² to4.0 g/m² body surface area, e.g., at least about 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or upto about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.5, 2.7, 3.0, or 3.5 g/m². In some embodiments, thecysteamine product may be administered at a total daily dose of about1-1.5 g/m² body surface area, or 0.5-1 g/m² body surface area, or about0.7-0.8 g/m² body surface area, or about 1.35 g/m² body surface area.Salts or esters of the same active ingredient may vary in molecularweight depending on the type and weight of the salt or ester moiety. Foradministration of the dosage form, e.g., a tablet or capsule or otheroral dosage form comprising the enterically coated cysteamine product, atotal weight in the range of approximately 100 mg to 1000 mg is used.The dosage form is orally administered to a patient suffering from fattyliver disease for which an cysteamine product would be indicated,including, but not limited to, NAFLD and NASH. Administration maycontinue for at least 3 months, 6 months, 9 months, 1 year, 2 years, ormore.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancer includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544, 1993).

The enterically coated cysteamine product can comprise variousexcipients, as is well known in the pharmaceutical art, provided suchexcipients do not exhibit a destabilizing effect on any components inthe composition. Thus, excipients such as binders, bulking agents,diluents, disintegrants, lubricants, fillers, carriers, and the like canbe combined with the cysteamine product. For solid compositions,diluents are typically necessary to increase the bulk of a tablet sothat a practical size is provided for compression. Suitable diluentsinclude dicalcium phosphate, calcium sulfate, lactose, cellulose,kaolin, mannitol, sodium chloride, dry starch and powdered sugar.Binders are used to impart cohesive qualities to a tablet formulation,and thus ensure that a tablet remains intact after compression. Suitablebinder materials include, but are not limited to, starch (including cornstarch and pregelatinized starch), gelatin, sugars (including sucrose,glucose, dextrose and lactose), polyethylene glycol, waxes, and naturaland synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, hydroxyethyl cellulose, and thelike), and Veegum. Lubricants are used to facilitate tablet manufacture;examples of suitable lubricants include, for example, magnesiumstearate, calcium stearate, and stearic acid, and are typically presentat no more than approximately 1 weight percent relative to tabletweight. Disintegrants are used to facilitate tablet disintegration or“breakup” after administration, and are generally starches, clays,celluloses, algins, gums or crosslinked polymers. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and the like. If desired, flavoring, coloring and/or sweeteningagents may be added as well. Other optional components for incorporationinto an oral formulation herein include, but are not limited to,preservatives, suspending agents, thickening agents, and the like.Fillers include, for example, insoluble materials such as silicondioxide, titanium oxide, alumina, talc, kaolin, powdered cellulose,microcrystalline cellulose, and the like, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride,sorbitol, and the like.

A pharmaceutical composition may also comprise a stabilizing agent suchas hydroxypropyl methylcellulose or polyvinylpyrrolidone, as disclosedin U.S. Pat. No. 4,301,146. Other stabilizing agents include, but arenot limited to, cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropyl methylcellulose phthalate, microcrystalline cellulose andcarboxymethylcellulose sodium; and vinyl polymers and copolymers such aspolyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonicacid copolymer, and ethylene-vinyl acetate copolymers. The stabilizingagent is present in an amount effective to provide the desiredstabilizing effect; generally, this means that the ratio of cysteamineproduct to the stabilizing agent is at least about 1:500 w/w, morecommonly about 1:99 w/w.

The tablets can be manufactured by first enterically coating thecysteamine product. A method for forming tablets herein is by directcompression of the powders containing the enterically coated cysteamineproduct, optionally in combination with diluents, binders, lubricants,disintegrants, colorants, stabilizers or the like. As an alternative todirect compression, compressed tablets can be prepared usingwet-granulation or dry-granulation processes. Tablets may also be moldedrather than compressed, starting with a moist material containing asuitable water-soluble lubricant.

In an alternative embodiment, the enterically coated cysteamine productare granulated and the granulation is compressed into a tablet or filledinto a capsule. Capsule materials may be either hard or soft, and aretypically sealed, such as with gelatin bands or the like. Tablets andcapsules for oral use will generally include one or more commonly usedexcipients as discussed herein.

For administration of the dosage form, i.e., the tablet or capsulecomprising the enterically coated cysteamine product, a total weight inthe range of approximately 100 mg to 1000 mg is used. The dosage form isorally administered to a patient suffering from a condition for which ancysteamine product would typically be indicated, including, but notlimited to, NAFLD and NASH.

The compositions of the disclosure can be used in combination with othertherapies useful for treating NAFL and NASH. For example, antioxidantssuch as glycyrrhizin, schisandra extract, ascorbic acid, glutathione,silymarin, lipoic acid, and d-alpha-tocopherol, and parenterallyadministering to the subject glycyrrhizin, ascorbic acid, glutathione,and vitamin B-complex may be administered in combination (eithersimultaneously in a single composition or in separate compositions).Alternatively, the combination of therapeutics can be administeredsequentially.

The effectiveness of a method or composition of the disclosure can beassessed by measuring fatty acid content and metabolism in the liver.Dosage adjustment and therapy can be made by a medical specialistdepending upon, for example, the severity of NAFL.

In addition, various prodrugs can be “activated” by use of theenterically coated cysteamine. Prodrugs are pharmacologically inert,they themselves do not work in the body, but once they have beenabsorbed, the prodrug decomposes. The prodrug approach has been usedsuccessfully in a number of therapeutic areas including antibiotics,antihistamines and ulcer treatments. The advantage of using prodrugs isthat the active agent is chemically camouflaged and no active agent isreleased until the drug has passed out of the gut and into the cells ofthe body. For example, a number of produgs use S—S bonds. Weak reducingagents, such as cysteamine, reduce these bonds and release the drug.Accordingly, the compositions of the disclosure are useful incombination with pro-drugs for timed release of the drug. In thisaspect, a pro-drug can be administered followed by administration of anenterically coated cysteamine compositions of the disclosure (at adesired time) to activate the pro-drug.

It is to be understood that while the disclosure has been described inconjunction with specific embodiments thereof, that the foregoingdescription as well as the examples which follow are intended toillustrate and not limit the scope of the disclosure. Other aspects,advantages and modifications within the scope of the disclosure will beapparent to those skilled in the art to which the disclosure.

EXAMPLES Example 1 Formulation of Enteric Coated Cysteamine

International Publication No. WO 2007/089670 described administration ofcysteamine to cystinosis patients using a nasoenteric tube to determinethe efficacy of enteric administration on the improvement in cystinosispatients. WO 2007/089670 showed that administration of cysteamineenterically improved the absorption rate of cysteamine and increasedplasma cysteamine levels. Enteric administration also reduced the levelsof cystine in leukocytes. These results showed that enteric cysteaminewas more efficacious than oral administration of cysteamine.

An enteric coated preparation on cysteamine (Cystagon-EC) was createdfor more efficacious and easier administration. CYSTAGON® capsules(Mylan Laboratories Inc., PA, USA) were enterically coated using a Model600 Wurster coating unit with a 4/6″ coating chamber. Coating materialis Eudragit® L30 D-55, Rohm GmbH & Co KG, Darmstadt, Germany) and the ECcompound was encapsulated (The Coating Place Inc, Verona, WI, Federalfacilities establishment number 2126906). Capsules were produced usingFDA approved facilities and materials.

Enteric coating was tested in vitro to verify the insolubility of thecapsules in gastric acid. Testing was done by placing the capsules into100 mL 0.1N HCL solution for 2 hours at 37° C. Capsules are consideredacceptable if less than 10% of the cysteamine is released. After 2 hoursthe pH of the solution is raised to pH 6.8 with NaHCO₃ buffer. Capsulesare considered acceptable if at least 80% of the cysteamine is releasedwithin 2 hours.

Six adult controls subjects and 6 patients with cystinosis have beenstudied using the Cystagon-EC. The plasma cysteamine levels were higherwhen the patient took Cystagon-EC than when they took the regularcysteamine (CYSTAGON®) preparation. In addition, when cystinosispatients took Cystagon-EC the 12 hour trough white cell cystine levelsremained at about <0.2 and usually below 1 nmol of half-cystine/mgprotein, suggesting that this new formulation of cysteamine is effectivewhen given twice daily.

Example 2 Administration of Cysteamine to Patients Suffering from FattyLiver Disease

Administration of cysteamine has been shown to relieve symptoms ofcystinosis by decreasing levels of damaging cystine. To determine theeffect of cysteamine on the fibrosis that causes liver damage in NASHpatients, an open-label, non-randomized, pilot study of 12 children andadolescents with non-alcoholic fatty disease treated with enteric-coatedcysteamine is performed.

Patients with an established diagnosis of NASH, who have undertakenlifestyle changes (such as diet and exercise) for at least three months,are used for the study. A full history and physical examination istaken. A symptom score devised for acid-peptic disease and previouslyused in children taking cysteamine is used. Blood is drawn for liverfunctions including hepatic transaminases, alkaline phosphatase,bilirubin and gamma-GT. Blood is also taken for complete blood count,ESR, CRP, fasting insulin and fasting lipid and cholesterol profile,markers of oxidative stress and of liver fibrosis (total 15 ml).Patients weight is recorded.

The study entry level for ALT is defined as ≧60 iu/L and a successfulresponse to therapy is normalization or >35% reduction in hepatictransaminase level. A normal ALT level is defined as 40 iu/L. Subjectsare started on enteric-coated cysteamine twice daily at a total dailydose of 1 g/m² body surface area with a maximum dose of 1000 mg twicedaily. Patients with cystinosis normally take 1-1.5 g/m² body surfacearea/day.

Any subject complaining of significant gastrointestinal symptoms mayhave his/her dose daily dose of cysteamine reduced by 10%. If GIsymptoms persist for 3 days despite a 10% decrease in dosage, thenfurther 10% decrements in dosage will permitted (to a maximum of 50% ofthe original dose). If symptoms persist despite the maximum decrease inEC-cysteamine dose, the subject is removed from the study.

If symptoms are severe, subjects may exit the study at any point. Ifpatients were on acid-suppression therapy such as proton-pumpinhibitors, they are asked to discontinue therapy one week beforecommencing EC-cysteamine. Patients are treated initially for 3 months,and for a maximum of 6 months, with EC-cysteamine. If a 10-25% reductionin hepatic transaminase levels are detected then treatment is extendedfor a further 3 months. If, however, there is <10% reduction of ALTlevel after 3 months therapy, the subject will take no further part inthe study. If there is an improvement in serum hepatic transaminaselevels (>35%) after six months of therapy, then patients are monitoredfor a further six months with a physical examination and the same bloodtests performed every two months. Menstruating females will undergo ablood pregnancy test at the start and every month during the study. Ifappropriate, patients are advised to take contraceptive precautionsusing double barrier method.

Patients are asked to maintain a diary of symptoms and will also be seenin the GCRC/clinic in order to obtain information with about are blindedto the identity of the patient's study drug. A symptom score devised foracid-peptic disease and previously used in children taking cysteamine isused. Every 4 weeks patients will have repeat blood tests includingliver function tests, complete blood count, and plasma cysteamine levels(10 ml). At the end of the study, patients will have all baselinelaboratory tests repeated.

Example 3

The effect of a cysteamine product was evaluated in a dietary animalmodel of non-alcoholic fatty liver disease (NAFLD), carried outgenerally as described in Otogawa et al., Am. J. Pathol., 170(3):967-980(2007). Male New Zealand white rabbits were fed a high-fat diet (HFD)containing 20% corn oil and 1.25% cholesterol in order to induceclinical and histological features characteristic of NAFLD andnon-alcoholic steatohepatitis (NASH). A pilot study of 7-days durationwas carried out using intraperitoneal (IP) dosing of cysteaminebitartrate on an every 8 hour schedule (Q8H) at two dose levels: 75 or250 mg/kg/day. A longer study of 8-weeks duration delivered cysteaminebitartrate in the drinking water at 25, 75 or 250 mg/kg/day.

As discussed in further detail below, the data from both studies showedthat cysteamine treatment produced an improvement in levels of hepatictransaminase (aspartate aminotransferase, or AST) compared to the HFDuntreated control group. AST elevation is considered one of the bestmarkers of liver inflammation in NAFLD and NASH. Compared to the HFDdiet control animals, AST levels were reduced by 1.6- to 1.9-fold withcysteamine treatment, i.e., reductions of 37 to 47%, respectively. Thedata also showed that cysteamine treatment was associated withbeneficial changes in LDH, a general marker of tissue damage, andbeneficial changes in lipid profile markers such as total cholesterol,LDL-cholesterol, and HDL-cholesterol, compared to the HFD controlgroups. The improvements observed in these rabbit models support theconclusion that cysteamine treatment of human non-alcoholic fatty liverdiseases (NAFLD) including NASH may confer clinical benefits.

Pilot Study.

In the pilot study, two different dose levels of cysteamine weredelivered by the intraperitoneal (IP) route every 8 hours (Q8H) for 7days to New Zealand white rabbits fed a high-fat diet (HFD). Malerabbits between 2.5 to 3.5 kg were divided into the following groups: 1)control standard diet, 2 animals, 2) control HFD, 2 animals, 3) low dosecysteamine bitartrate, 75 mg/kg/day, HFD, 4 animals, and 4) high dosecysteamine bitartrate, 250 mg/kg/day, HFD, 4 animals. Endpoints includeddaily standard clinical observations, quantitative daily foodconsumption, body weights on Study Day (SD) −1, 2, 5, and 8 (day ofnecropsy), and blood samples collected on SD-1 and SD8 for theevaluation of selected clinical chemistries (alanine aminotransferase(ALT), aspartate aminotransferase (AST), amylase, lipase, totalcholesterol, triglycerides, lactate dehydrogenase (LDH), high densitylipoprotein cholesterol (HDL-cholesterol), and low density lipoproteincholesterol (LDL-cholesterol)) and a full hematology panel. Animals weresacrificed on SD8.

No pharmacologically important differences were found regarding theclinical observations, the body weights, or hematology values obtainedat baseline on SD-1 and SD8. The values observed for ALT, amylase,lipase, triglycerides, and HDL-cholesterol were not different betweenthe groups over the 7 days of the study. However, increases wereobserved in total cholesterol, LDL-cholesterol and LDH values in thoseanimals fed the HFD.

Importantly, compared to the HFD control group, the groups treated withcysteamine showed improvements in four serum chemistry values: AST,total cholesterol, LDL-cholesterol, and LDH.

AST has emerged as the best marker of liver inflammation in NASH and isconsidered to be a superior marker to ALT. Compared to the HFD controlgroup, a decrease was observed in the mean AST values on SD8 in the highdose cysteamine group (250 mg/kg/day), as shown in FIG. 1. The mean ASTvalue for the control HFD group was 19.0 U/L, whereas the rabbits thatreceived 250 mg/kg/day cysteamine showed a 1.9-fold decrease in thisvalue to 10.0 U/L, or only 47% of the control HFD value. Because therewere only 2 animals in the control HFD group, it was not possible tomake statistical comparisons. However, comparison of the AST results ofthe 75 mg/kg/day group on SD8 against those of the 250 mg/kg/daycysteamine animals indicated that the high dose cysteamine group wasstatistically different from the low dose group by the Mann-Whitney Utest, p=0.03. These data showed that cysteamine treatment at 250mg/kg/day on this regimen had a positive impact on AST values.

Mean serum total cholesterol values at baseline (SD-1) ranged from 42.5to 55.25 mg/dL across all groups, which is within the lab's historicalrange for normal rabbits of 20-78 mg/dL. On SD8, rabbits who receivedcysteamine at either 75 or 250 mg/kg/day were found to have less of anincrease in mean total cholesterol as compared to the control HFD group,as shown in FIG. 2. The control rabbits in Group 2 on the HFD had a meantotal cholesterol value of 842 mg/dL on SD8, approximately a 20-foldincrease over their baseline values. The Group 3 rabbits, who received75 mg/kg/day cysteamine, had a mean value of 652 mg/dL, only about a12-fold increase over their baseline value, or 23% less of an increasethan the HFD controls. The rabbits in Group 4, who received 250mg/kg/day cysteamine, had a mean value of 347 mg/dL on SD8, only about a7.5-fold increase over their baseline value, or 59% less of an increasethan the HFD control values. These data showed that cysteamine treatmentresulted in a clear dose-dependent reduction in the serum totalcholesterol increase due to the HFD diet.

LDL-cholesterol values also appeared to be impacted by cysteaminedosing. As seen with the total cholesterol marker, the increase inLDL-cholesterol observed in the control HFD group was noticeablylessened in those rabbits treated with cysteamine at either 75 or 250mg/kg/day, as shown in FIG. 3. The mean LDL-cholesterol value across allgroups at baseline ranged from 9.5 to 18 mg/dL, within the lab'shistorical range of 4 to 19 mg/dL. On SD8, the control HFD rabbits had amean value of 272.5 mg/dL, an increase of about 29-fold over baseline.The rabbits in Group 3 treated with 75 mg/kg/day cysteamine had a meanvalue of 210 mg/dL on SD8, an increase of only about 12-fold compared totheir respective baseline values, or 23% less of an increase than theHFD controls. The rabbits in Group 4 treated with 250 mg/kg/day had amean LDL-cholesterol value of 150.5 mg/dL, an increase of only about14-fold compared to their baseline values, or 45% less of an increasethan the HFD controls. These data showed that cysteamine treatmentresulted in notable reductions in the LDL-cholesterol increases due tothe HFD diet.

LDH values showed a similar trend: rabbits that received cysteamine ateither 75 or 250 mg/kg/day had less of an increase in LDH than thecontrol HFD rabbits, as shown in FIG. 4. The control HFD rabbits inGroup 2 had a mean LDH value of 190 U/L on SD8, an increase of 1.7-foldover their baseline values. The rabbits in Group 3 (75 mg/kg/day) hadmean values of 128 U/L on SD8, which was a decrease of 1.2-fold comparedto their baseline values, or 33% of the control HFD values on SD8. Therabbits in Group 4 (250 mg/kg/day) had mean values of 77.5 U/L on SD8,which was a decrease of 3.1-fold compared to their baseline values, or59% of the control HFD values on SD8. These data showed that cysteaminetreatment resulted in a dose dependent reduction of these LDH values ascompared to the control rabbits fed the HFD.

The data show that treatment of rabbits fed a HFD with cysteaminebitartrate at either 75 or 250 mg/kg/day IP on a Q8H schedule resultedin an improvement in levels of liver transaminase (AST), an importantmarker of liver inflammation and damage in NAFLD. Cysteamine treatmentwas also associated with beneficial changes in the biochemical serummarkers total cholesterol, LDL-cholesterol, and LDH. Taken together, thedata support the conclusions that cysteamine treatment may conferclinical benefits in human patients with NAFLD such as NASH.

8-Week Study.

The purpose of this study was to evaluate the effects of cysteaminetreatment in an animal model of NAFLD and NASH in which male New Zealandwhite rabbits are fed a high fat diet (HFD) containing 20% corn oil and1.25% cholesterol to produce clinical and histological featurescharacteristic of NAFLD and NASH.

The study design included five groups of eight rabbits. Two controlgroups did not receive cysteamine in their drinking water: a controlgroup that was fed conventional rabbit chow, and another control groupthat was fed the HFD. In three groups of rabbits fed the HFD, cysteaminebitartrate was introduced in the drinking water at concentrationscalculated to deliver either 25, 75, or 250 mg/kg/day. Drinking waterwas prepared fresh daily based on room temperature stability informationfor cysteamine bitartrate across this concentration range.

Observations during the study included twice weekly body weights, dailyfood and water consumption, and daily clinical observations. Bloodsamples were collected prior to the start of the study, and on weeks 2,4, 6, and 8 for a full panel of hematology parameters and a selectedpanel of serum chemistries, including ALT, AST, amylase, lipase, totalcholesterol, triglycerides, LDH, HDL-cholesterol, and LDL-cholesterol.

In all groups that received the HFD, clinical observations such as roughcoats and quiet behavior began to be observed around the middle of week8, while signs of jaundice began to appear earlier, in the middle ofweek 6. Some animals displayed dark or red colored urine in these sametimeframes, suggesting possible bile blockages (cholestasis). Throughoutthe study, animals on the HFD much more frequently exhibited soft stoolscompared to the animals on the standard diet. Animals on the HFD alsoexhibited histological features suggestive of NAFLD. Three animals inGroup 4, the mid-dose cysteamine group, died on study or were sacrificedmoribund on SD 51, 55, and 56. One animal in Group 5, the high dosegroup, was sacrificed moribund on SD 55. These deaths on study appearedto be associated with advanced NAFLD.

The body weight data showed that animals on the standard diet and theHFD gained weight at approximately the same rate during the first 6weeks of the study. However, beginning by week 7, the animals on the HFDbegan to lose weight compared to the standard diet. The body weights ofthe cysteamine treated animals seemed to parallel those of the controlHFD animals. The food consumption data showed that the animals fed theHFD consumed less food than those on the standard diet after the firstweek of the study, which would be expected due to the higher caloriccontent of the HFD. The food consumption of the treated groups and theHFD control group was similar. By around week 6, animals on the HFD wereconsuming only about 15 to 30% of the amount of food consumed by animalson the standard diet based on the area under the curve (AUC).

The water consumption data followed a similar pattern. All animals onthe HFD consumed less water than those on the standard diet, presumablydue to a higher moisture content in the HFD. Based on AUCs, the controlHFD group consumed about 65% of the water consumed by the animals on thestandard diet. The groups that drank cysteamine-containing waterconsumed about two-thirds of the water consumed by the HFD controlgroup. These data would suggest that the cysteamine-containing water mayhave been somewhat less palatable than the control water to theserabbits.

The hematology data did not reveal pharmacologically important changesacross the study. There was a trend to slightly increased white bloodcell (WBC) counts in the control rabbits fed the HFD compared to thestandard diet controls, primarily due to lymphocytes. Thecysteamine-treated groups were similar to the HFD control group.

The serum chemistry data reflected differences in the control animalsfed the HFD compared to the standard diet control animals. At the end ofthe study (week 8), the HFD control animals in Group 2 had increases inAST (2.6-fold), lipase (6.6-fold), cholesterol (64-fold), triglyceride(3.8-fold), LDH (3-fold), HDL-cholesterol (2.3-fold), andLDL-cholesterol (55-fold) compared to the standard diet control values(Group 1). Amylase and ALT values were unchanged.

AST is considered to be a better marker of hepatic inflammation thanALT. At 8 weeks, control animals fed the HFD had a mean AST value of117.1 U/L, a 2.6-fold increase compared to control animals fed astandard diet, as shown in FIG. 5. However, the mean AST values in boththe low-dose (25 mg/kg/day) and the high-dose (250 mg/kg/day) cysteaminetreatment groups were decreased compared to the control HFD animals. TheGroup 3 animals (25 mg/kg/day) had a mean AST value of only 62.5 U/L, a1.9-fold decrease relative to the HFD control group, a reduction of 47%.Similarly, the Group 5 animals (250 mg/kg/day) had a mean AST value ofonly 75.7 U/L, a 1.6-fold decrease relative to HFD controls, a reductionof 35%. Given the difficulties in assessing drug delivery by supplyingcysteamine in the drinking water, it is notable that these decreases inAST values were associated with two out of the three cysteaminetreatment groups. Similar decreases in AST were also found in the pilotstudy.

As observed in the pilot study, decreases in LDH were also observed inthis study in animals treated with cysteamine. As shown in FIG. 6, atweek 8 the mean LDH value was 375 U/L in the Group 2 HFD controlanimals. Exposure to cysteamine resulted in a decrease in LDH values atall three dose levels in the treated animals compared to the Group 2 HFDcontrols. In Group 5, the high dose (250 mg/kg/day) group, the mean LDHvalue at week 8 was 140 U/L, nearly identical to the Group 1 standarddiet control group mean LDH value of 125.6 U/L. This difference betweenthe HFD control LDH value and the high-dose cysteamine (250 mg/kg/day)value was statistically significant by the Mann-Whitney U test, p=0.03.These data showed that cysteamine treatment markedly reduced theincrease in LDH caused by the HFD.

It is well-recognized that HDL-cholesterol levels are positiveindicators of healthy lipid profiles. Rabbits are known to be aparticularly good model for human lipid profiles because they havebaseline ratios similar to those found in humans, and they areconsidered a good model of human cardiovascular disease. Therefore, itwas notable that in this study, animals treated with the high-dosecysteamine (250 mg/kg/day) showed a beneficial increase inHDL-cholesterol compared to both the standard diet control group as wellas the HFD control group, as shown in FIG. 7. At week 8, the meanHDL-cholesterol value in the 250 mg/kg/day cysteamine group was 58.3mg/dL, a 1.6-fold increase over the HFD control mean value of 36.9mg/dL, and a 3.7-fold increase over the control standard diet value of15.7 mg/dL.

Taken together, the data collected in this 8-week study showed thatrabbits fed the HFD developed clinical and serological featuresassociated with liver disease consistent with NAFLD and NASH. Cysteaminedosing in the water bottles likely resulted in variable delivery of thedrug to the treated animals. Nonetheless, it was found that two of thesame serum chemistry markers that were improved in the pilot study werealso improved in this 8-week study in the presence of cysteamine: ASTand LDH. These were considered important findings given that AST isconsidered to be the best marker of inflammation in human NASH and thatreductions in LDH probably also reflect less inflammation and likelyprotection from cytotoxicity in these animals.

Another notable finding in this longer term study was that cysteaminetreatment was associated with a beneficial rise in the serumHDL-cholesterol levels.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

1. A method of treating a patient suffering from non-alcoholic fattyliver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) comprisingadministering a composition comprising a therapeutically effectiveamount of cysteamine or a pharmaceutically acceptable salt thereof, orcystamine or a pharmaceutically acceptable salt thereof, wherein thecomposition is enterically coated. 2-3. (canceled)
 4. The method ofclaim 1, wherein the total daily dose of cysteamine or apharmaceutically acceptable salt thereof, or cystamine or apharmaceutically acceptable salt thereof is about 0.5-2.0 g/m².
 5. Themethod of claim 1, wherein the total daily dose of cysteamine or apharmaceutically acceptable salt thereof, or cystamine or apharmaceutically acceptable salt thereof is about 0.5-1.0 g/m².
 6. Themethod of claim 1, wherein the cysteamine or a pharmaceuticallyacceptable salt thereof, or cystamine or a pharmaceutically acceptablesalt thereof is administered at a frequency of 4 or less times per day.7. The method of claim 6, wherein the cysteamine or a pharmaceuticallyacceptable salt thereof, or cystamine or a pharmaceutically acceptablesalt thereof is administered two times per day.
 8. The method of claim1, wherein the composition provides increased delivery of the cysteamineor a pharmaceutically acceptable salt thereof, or cystamine or apharmaceutically acceptable salt thereof to the small intestine. 9-11.(canceled)
 12. The method of claim 1, wherein the composition comprisesan enteric coating that releases the cysteamine or a pharmaceuticallyacceptable salt thereof, or cystamine or a pharmaceutically acceptablesalt thereof when the cysteamine or a pharmaceutically acceptable saltthereof, or cystamine or a pharmaceutically acceptable salt thereofreaches the small intestine or a region of the gastrointestinal tract ofa subject in which the pH is greater than about pH 4.5.
 13. The methodof claim 12, wherein the composition comprises a coating selected fromthe group consisting of polymerized gelatin, shellac, methacrylic acidcopolymer type C NF, cellulose butyrate phthalate, cellulose hydrogenphthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate(PVAP), cellulose acetate phthalate (CAP), cellulose acetatetrimellitate (CAT), hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulosesuccinate, carboxymethyl ethylcellulose (CMEC), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and acrylic acid polymersand copolymers, typically formed from methyl acrylate, ethyl acrylate,methyl methacrylate and/or ethyl methacrylate with copolymers of acrylicand methacrylic acid esters.
 14. The method of claim 1, wherein thecomposition is administered orally.
 15. (canceled)
 16. The method ofclaim 1, wherein the administering results in improvement in liverfibrosis compared to levels before administration of the composition.17. The method of claim 1, wherein the administering results in areduction in fat content of liver.
 18. The method of claim 1, whereinthe administering results in a reduction in the incidence of orprogression of cirrhosis.
 19. The method of claim 1, wherein theadministering results in a reduction in the incidence of hepatocellularcarcinoma.
 20. The method of claim 1, wherein the administering resultsin a decrease in hepatic aminotransferase levels compared to levelsbefore administration of the composition.
 21. The method of claim 1,wherein the administering results in a reduction in hepatic transaminaseof between approximately 10% to 40% compared to levels before treatment.22. The method of claim 1, wherein the administering results in areduction in alanine anminotransferase levels in a treated patient toapproximately 30%, 20% or 10% above normal ALT levels, or at normal ALTlevels (≧40 iu/L).
 23. The method of claim 1, wherein the administeringresults in a reduction in aspartate anminotransferase levels in atreated patient to approximately 30%, 20% or 10% above normal AST levelsor to normal AST levels.
 24. The method of claim 1, wherein theadministering results in a reduction in serum ferritin levels comparedto levels before treatment with the composition.
 25. The method of claim1, wherein the cysteamine or a pharmaceutically acceptable salt thereof,or cystamine or a pharmaceutically acceptable salt thereof isadministered with a second agent useful to treat NAFLD or NASH.
 26. Themethod of claim 1, wherein the patient is a child or adolescent.
 27. Themethod of claim 1, wherein the composition increases delivery to theproximal or mid-small intestine or both.
 28. The method of claim 1,wherein the composition increases delivery to one or more of theduodenum, jejunum or mid-ileum.
 29. The method of claim 13, wherein thecoating material begins to dissolve in an aqueous solution at pH betweenabout 4.5 to about 5.5.
 30. The method of claim 1, wherein thecomposition is formulated as a tablet or capsule.
 31. The method ofclaim 31, wherein each dose of cysteamine or cystamine orpharmaceutically acceptable salt thereof is about 0.5-1.0 g/m².
 32. Themethod of claim 1, wherein the composition comprises cysteamine or apharmaceutically acceptable salt thereof.
 33. The method of claim 1,wherein the cysteamine salt is cysteamine bitartrate.